Mutagenesis in vitro by DNA polymerase from an RNA tumour virus

Abstract

The fidelity of DNA synthesis has usually been determined from polynucleotide templates of restricted base composition. Mis-matched nucleotides were simply identified as those non-complementary to the template. Error rates estimated in this way are less than were predicted on the basis of Watson-Crick base pairing, 10^-2 (refs 4-6), but greater than were inferred from spontaneous mutation frequencies, 10^-8-10^-11 (ref. 7). However, in vitro error rates measured for the same enzyme vary from one synthetic template to another. In addition, the repeating structure of homopolymers and copolymers may, by slippage and looping-out of mis-matched bases, allow a higher frequency of mis-pairing than natural DNA of random sequence. To avoid these problems, we have recently developed a method for measuring the accuracy of in vitro DNA synthesis using a natural DNA template. We now report that a DNA polymerase which has a high frequency of mis-incorporation with synthetic polynucleotides1 is also mutagenic when copying natural DNA in vitro.